Supelco Reporter Vol. 27 - Sigma-Aldrich

ReporterVolume 27.4

During the last decade, analysis of food and beverage products for both harmful and beneficial compounds has become a topic of major interest.

Food & Beverage Analysis

•• Liquid Chromatography

•• Sample Handling

•• Gas Chromatography

•• Standards

•• Accessories

•• Chiral Chromatography

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Volume 27.4

Reporter Visit us on the web at sigma-aldrich.com/thereporter

Reporter is published 5 times a year by Supelco Marketing, 595 North Harrison Road, Bellefonte, PA 16823-0048.

Innovation & Trends in Sample Preparation

An Trinh

Product Manager, Sample Prep

Volume 27.4

Table of ContentsLiquid Chromatography

UHPLC with the new Ascentis® Express Phenyl-Hexyl Column .........................3

NEW! Ultra-High Performance Liquid Chromatography Accessories .............7

Reversed-Phase HPLC Buffers.............8

Discovery® BIO GFC 3 μm Packings ..10

Sample HandlingExtraction of Nitroimidazoles from Milk and Eggs using Molecularly Imprinted Polymers ..........................15

The Analysis of Resveratrol in Red Wine by On-Fiber Derivatization/SPME .........................18

Gas ChromatographyComparing the Enantioselectivity of Cyclodextrin-Based GC Chiral Stationary Phases .............................11

Determination of Triglycerides and Waxes in Food Products Using Cool On-Column Injection and the MET-Biodiesel Capillary Column .......12

StandardsNSI Solutions Inorganic Product Line ....................................20

Characterized Reference Oils for Standardizing Lipid Procedures ........22

Mono-, Di-, and Triglyceride Chemical Standards. ........................22

AccessoriesSGE Slim Line Syringes for CTC/Leap Autosamplers ...................23

Accelerating Customers’ Success through Innovation and Leadership in Life Science, High Technology and Service

Dear Colleague:

As LC-MS becomes increasingly commonplace, analysts are expected to develop assays that are faster, provide greater throughput, and achieve sensitivity levels that were near impossible just a decade prior.

I highlight below some of the recent trends observed in sample prep technology:

Multi-Residue Methods. Most solid phase extraction procedures target a specifi c analyte or class of analytes; however, many researchers, especially in the fi eld of food safety/quality, must monitor for dozens of classes of analytes. In such applications, fi nding a single SPE condition that can adequately retain such a broad range of compounds would be impossible. To negotiate this issue, researchers have developed SPE techniques that target the retention and removal of specifi c interferences for a given sample matrix. These researchers have recognized that if the sample prep method can remove one to three key interferences, targeted LLODS can often be achieved. A classic example is the recently popular QuEChERS technique for multi-residue pesticide analysis.

Simple and Generic Methods. In order to maximize the throughput benefi ts of LC-MS technology, most researchers desire faster and easier sample prep. As a result, simple and generic techniques such as protein precipitation have become the preferred method when analyzing biological plasma. When improved selectivity is desired, such analysts look towards SPE phases that are more amenable to generic methodology such as hydrophilic polymer SPE phases and mixed-mode SPE technology.

Highly Selective Techniques. As mentioned previously, more and more assays are targeting lower limits of detection. As trace analysis becomes mainstream, highly selective sample prep techniques are becoming more prevalent. For example, molecularly imprinted polymers have seen a surge in publications in recent years; and many of the more recent and exciting developments in sample prep technology are application specifi c. In the last 5 years, Supelco has commercialized an array of application specifi c products including: HybridSPE™-Precipitation technology for protein and phospholipid removal, SupelMIP™ molecularly imprinted polymers, silver-ion SPE for FAMEs analysis, Supelclean™ sulfoxide SPE for PCB fractionation... to name a few.

To learn more about these trends and our solutions for them, visit our web site at sigma-aldrich.com/sampleprep or call our Technical Service Group at 800-359-3041 (US and Canada only) and 814-359-3041.

Best regards,

An TrinhProduct Manager, Sample Prep

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Volume 27.4

Richard A. Henry(2) and Hillel K. [email protected]

Peak resolution is a requirement for sensitive, accurate

chromatographic analysis. Scientifi c meetings and

publications have been dominated recently by reports on

HPLC columns with Fused-Core™ particles and porous

particles in the sub-2 μm range, which can both signifi -

cantly enhance resolution and speed by producing either

higher effi ciency (N) for the same column length or

equivalent effi ciency with shorter column length.

Ultra-high performance liquid chromatography (UHPLC)

comes at a price of much higher pressure when sub-2 μm

particles are employed. The revolutionary Ascentis Express

2.7 μm Fused-Core silica particle (Figure 1) has quickly

become accepted as an attractive alternative because it is

equivalent in performance to particles in the sub-2 μm

range. With a very narrow particle size distribution,

Ascentis Express columns employ conventional 2 μm frits

and operate ruggedly at much lower pressures that are

within the comfort zone of conventional HPLC instruments.

column family, and describes how column selectivity and

higher effi ciency can be coupled to achieve much faster

separations than have previously been possible.

The vast majority of UHPLC separations have been

carried out with C18 columns in the classic reversed-phase

(RP) mode; however, suppliers now offer many different

phases. Although no one would dispute the fact that

UHPLC columns with different phases are needed, very

little has been published yet on the performance that can

be expected from UHPLC columns having different,

complementary selectivity to C18 and C8. Two of the

most popular polar-RP phases are RP-Amide, which is

often categorized as an embedded polar-group phase,

and Phenyl, which can interact with solutes by π-π

mechanisms. A brief retention and selectivity comparison

for the Ascentis Express column family is given in Table 1.

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Ultra-High Performance Liquid Chromatography (UHPLC) with the new Ascentis Express Phenyl-Hexyl Column

0.5

μm

1.7

μm

2.7

μm

G004850

Figure 1. Fused-Core Structure of Ascentis Express

C18 and C8 phases are highly popular because they are

stable, reproducible, and easy-to-use. Retention correlates

closely with log P values, which have been established for

many solutes. Solute ionization causes retention to

decrease in a predictable manner and is relatively easy to

control by adding dilute acids, bases, and buffers to the

mobile phase. Changing the organic component of the

mobile phase between acetonitrile and methanol (or other

solvents) allows the user to tweak resolution because

solvation affects phase structure and selectivity. Tempera-

ture is also a useful variable for optimizing phase selectiv-

ity. Columns with C18 and C8 phases will frequently give

optimum resolution when solutes are nonpolar or slightly

polar; however, columns with polar-RP phases such as

RP-Amide or Phenyl-Hexyl will often show improved (continued on page 4)

Table 1. Brief Overview of Ascentis Express Column Retention and Selectivity

Ascentis Express Principle PrincipleFused-Core Retention SolutePhase Mode Interaction

C18 Reversed-Phase (RP) Hydrophobic (dispersive)

C8 Reversed-Phase (RP) Hydrophobic (dispersive)

RP-Amide RP with embedded Hydrophobic and polarity H-bonding

Phenyl-Hexyl RP with pendant Hydrophobic and π-π aromaticity

HILIC (Silica) HILIC (or normal Hydrophilic (dipole, phase) H-bonding, ion exchange)

Why Do We Need Different HPLC Phases?

Even though new column technologies have more than

doubled the plates per meter possible with traditional 5 μm

columns, resolution still cannot be routinely achieved in

every case without the ability to adjust retention and

selectivity by proper selection of column stationary and

mobile phases. This article features Ascentis Express

Phenyl-Hexyl phase, a new addition to the Fused-Core

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retention and selectivity for more polar solutes. It should

be emphasized that even polar-RP phases have a signifi -

cant alkyl phase character in addition to their polar

character. The same mobile phase solvents and techniques

may be employed with polar-RP phases, with comparable

phase stability to C18.

The RP-Amide phase is complementary to C18 because

the amide group has several unique features: 1) strong

interaction by H-bonding when solutes can donate or

accept protons, 2) effective shielding of silanols by internal

H-bonding between amide group and silica surface, and

3) the ability to wet and operate well, even in 100%

aqueous solvents. H-bonding allows solutes with carboxyl

and phenol groups to be retained much longer and

separate much better on RP-Amide than on C18 or C8.

Shielding prevents solutes with amino groups from

interacting with silanols and can result in shorter retention

and sharper peaks on amide phases. Another interesting

feature of amide phases is that methanol and other

alcohols become much stronger solvents when H-bonding

between phase and solute occurs. Except for the special

situations listed above, an RP-Amide phase often performs

similar to C18 due to the long alkyl chain extending away

from the surface.

The Phenyl phase has unique selectivity arising from

solute interaction with the aromatic ring and its delocalized

electrons. It is complementary (orthogonal) to both C18

and RP-Amide phases because of this unique aromaticity.

An unsubstituted phenyl ring is a π-donor or Lewis base,

which interacts strongly with π-acceptors and any electron-

defi cient Lewis acid. Phenyl phases also tend to exhibit

good shape selectivity, which may originate from solute

multipoint interaction with the planar ring system. More

retention and selectivity will often be observed for solutes

with aromatic electron-withdrawing groups (fl uorine, nitro,

etc.) or with a delocalized heterocyclic ring system such as

the benzodiazepine compounds studied.

UHPLC Results with Ascentis Express Phenyl-Hexyl

Low-pressure drop with high effi ciency and a fl at van

Deemter curve have been confi rmed for Phenyl-Hexyl, as

shown in Figure 2. In general, more than twice the

column effi ciency of 5 μm particles can be expected for all

Ascentis Express Fused-Core columns at pressures that are

easily managed with all HPLC instruments. Note that

20,000 plates have been achieved for a 10 cm x 3 mm I.D.

column operating at optimum fl ow. A Jasco X-LC HPLC

instrument was used for the study. As shown in Figure 3,

(continued from page 3)

the selectivity of Ascentis Express Phenyl-Hexyl is very

similar to that of other commercial Phenyl columns, so

methods can be readily transferred between columns.

The difference in effi ciency and pressure drop for the two

porous 3 μm columns can be explained by different

particle size distributions.

Figures 4-6 show comparisons of fi ve benzodiazepines

separated on the four Ascentis Express RP phases in

water:acetonitrile and water:methanol mobile phases. No

additives were employed in order to observe the interaction

between these polar solutes and the different phases;

however, a dilute buffer will normally be used for develop-

ment of a validated method. The addition of 10-20 mM

buffer at neutral pH typically has little or no effect upon the

separation with these highly deactivated column phases.

Note that overall retention in acetonitrile is similar for

the four bonded phases, but elution order is different. The

two less polar compounds, diazepam and desmethyldiaz-

epam, elute late and show the same order for all columns

due to predominance of hydrophobic interactions. The

more polar solutes, however, elute earlier and interact

differently with Phenyl-Hexyl and the other phases. With

this test sample and operating conditions, three of the

four Ascentis Express RP columns provide good resolution

with different selectivity; however, Phenyl-Hexyl shows

the best retention and selectivity.

Figure 2. Flow Performance of Ascentis Express Phenyl-Hexyl Column with Neutral Probes10 cm x 3 mm I.D. column, water:acetonitrile, 50:50, 35 °C, 250 nm, 2 μL

Effi

cien

cy, N

22,000

20,000

18,000

16,000

14,000

12,000

10,0000.0 0.5 1.0 1.5

mL/min

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eigh

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3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.00 1 2 3 4 5 6 7

mm/sec

naphthalene, k = 4.4

naphthalene, k = 4.4

G004823

G004824

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Figure 3. Comparison of Phenyl Column Selectivity for Benzodiazepines

column: as indicated - 10 cm x 3 mm I.D. mobile phase A: water mobile phase B: acetonitrile mobile phase ratios: A:B = 65:35 fl ow rate: 0.6 mL/min. temp.: 35°C det.: UV, 250 nm injection: 2 μL

Sample (0.1 g/L ea.) 1. Oxazepam 2. Alprazolam 3. Clonazepam 4. N-desmethyldiazepam 5. Diazepam

0 2 4 6 8 10Min

G004825

G004826

G004827

Figure 4. Benzodiazepine Structures

N = 18,900

k = 10.4

N = 14,200

k = 14.1

N = 10,600

k = 13.3

Ascentis Express Phenyl-Hexyl

Total = 3000 psiCol = 2360 psi

Column A Phenyl-Hexyl, 3μ


Column B Phenyl, 3μ


Diazepam N-desmethyldiazepam Alprazolam

Clonazepam Oxazepam

G002881 G003672 G003670

G002879 G003654

(continued on page 6)

Figure 5. Benzodiazepines in 35% Acetonitrile Mobile Phase with no Additive

column: Ascentis Express, as indicated - 10 cm x 3 mm I.D., 2.7 μm particles mobile phase A: water mobile phase B: acetonitrile mobile phase ratios: A:B = 65:35 fl ow rate: 0.6 mL/min. pressure: 3000 psi total, 2360 psi column temp.: 35 °C det.: UV, 250 nm injection: 2 μL

1

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Phenyl-Hexyl

C18

C8


A switch to water:methanol in Figure 6 (page 6) shows a

dramatic change in retention for Ascentis Express Phenyl-

Hexyl. In water:methanol mobile phase, the phenyl group

interacts much more strongly than the other phases with

the solute heterocyclic ring system, presumably by a π-π

mechanism. Kazakevitch (1) has published evidence that

methanol forms only monolayer coverage on aromatic

phases (and also thinly solvates other phases), which allows

the aromatic selectivity to shine through more strongly.

Elution order for the polar compounds also changes from

that of water:acetonitrile conditions. For this test sample,

Ascentis Express Phenyl-Hexyl selectivity is clearly superior

in water:methanol to the other phases.

G004809

G004808

G004807

G004806

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Featured Products+Ascentis Express Phenyl-Hexyl HPLC Columns

Length (cm) I.D. (mm) Cat. No.

3 2.1 53332-U 5 2.1 53334-U 7.5 2.1 53335-U 10 2.1 53336-U 15 2.1 53338-U 3 3.0 53341-U 5 3.0 53342-U 7.5 3.0 53343-U 10 3.0 53345-U 15 3.0 53346-U 3 4.6 53347-U 5 4.6 53348-U 7.5 4.6 53351-U 10 4.6 53352-U 15 4.6 53353-U

Figure 7. Ascentis Express Phenyl-Hexyl High-Speed Separation

column: 10 cm x 3 mm I.D. mobile phase A: water mobile phase B: acetonitrile mobile phase ratios: A:B = 50:50 fl ow rate: 1.2 mL/min pressure: 5525 psi temp.: 35 °C det.: UV, 250 nm injection: 2 μL

Figure 6. Benzodiazepines in 60% Methanol Mobile Phase with no Additive

column: Ascentis Express, as indicated - 10 cm x 3 mm I.D. mobile phase A: water mobile phase B: methanol mobile phase ratios: A:B = 40:60 fl ow rate: 0.6 mL/min pressure: 4200 psi total; 3420 psi column temp.: 35 °C det.: UV, 250 nm injection: 2 μL

A major objective for development of UHPLC columns is

to gain separation speed and sample throughput by using

shorter columns at higher fl ow rates. These columns also

improve sensitivity and uses less solvent. The fl at van

Deemter curve shown in Figure 2 allows an increase in

fl ow rate and mobile phase velocity without a signifi cant

loss in resolution. This result is illustrated in Figure 7 for a

neutral polar test mix in water:acetonitrile mobile phase.


Phenyl-Hexyl

C8

C18

RP-Amide


Conclusions

A new Phenyl-Hexyl phase has been paired with Fused-

Core particles to complete the primary Ascentis Express

column family. High performance with lower pressure drop

than other UHPLC columns has been confi rmed for all

Fused-Core particle phases. Ascentis Express Phenyl-Hexyl

correlates well to other Phenyl phases for easy method

development or method transfer. Selectivity for benzodiaz-

epine compounds has been compared to the other Ascentis

Express RP phases in water:acetonitrile and water:methanol.

The extra retention possible with Phenyl phases in

water:methanol has been demonstrated for these heterocy-

clic aromatic compounds. The potential for faster, more

sensitive assays using Ascentis Express Phenyl-Hexyl and all

Ascentis Express phases has been shown.

References 1. Kazakevitch, Y. V., et al. J. Chromatogr., A. 2005, 1082, 158–165.

2. Consultant to Supelco/Sigma-Aldrich.

0 2 4 6 8Min G004831

31

2

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5

3 1,2

4

5

3 1

2

4

5

31

24

5

0 1.0 2.0Min G004832

G004830

G004829

G004828

N = 19,200

1. Uracil 2. Acetophenone 3. Benzene 4. Toluene 5. Naphthalene

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NEW! Ultra-High Performance Liquid Chromatography Accessories

Ultra-high performance liquid chromatogra-

phy (UHPLC) columns containing particles that

are smaller than 3 micron have created

very narrow peak widths that

can no longer be routinely

measured by all HPLC

systems. The effi ciency of

separations performed with low-

volume columns is highly dependent

on the system having compo-

nents designed to minimize

band-width. With UHPLC

systems it is good laboratory

practice to install the proper

fi ttings, ferrules, and other

accessories to ensure the

analytical results show no extra

column effects created by improp-

erly assembled accessories. Typical,

more popular PEEK and stainless steel

fi ttings, pre-column fi lters,

guard columns, and tubing,

must be reliable enough to

hold at the pressures needed

(200 bar) with today’s UHPLC

instrumentation.

Upgrading the UHPLC system can bring on

many challenges due to the vast array of

commercially available products. To assist in

achieving the system and pressure requirements,

Supelco has introduced a line of UHPLC

accessories from the most trusted names in the

industry into this rapidly growing area of

chromatography making the product selection

process easier. This newly expanded line of

accessories for low volume and high sensitivity

analytical applications helps to maximize the

effi ciency of your analysis and protect your

column investment. These new products

compliment the vast array of HPLC accessory

products we currently offer.

For assistance with new product selection,

please contact our Technical Service group at

[email protected] or visit us on the web at

sigma-aldrich.com/hplc

are smaller

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Featured Products+Description Pkg. Size Cat. No.

Pre-Column Filters

2 μm Solvent Filter Assembly with frit (1.4 μL swept volume) 1 51231-U0.5 μm Solvent Filter Assembly with frit (1.3 μL swept volume) 1 51232-U0.5 μm Solvent Filter Assembly with frit ( 0.84 μL swept volume) 1 51233-UMini Microfi lter Assembly

1 μm, SS Frit 1 51242-U1 μm, Titanium Frit 1 51243-UUltra-High Performance Fittings for 1/32 in. Tubing

PEEK LiteTouch® Nut (Black) 10-32 10 51256-UPEEK LiteTouch® Micro Ferrule (Black) 1/32 in. 10 51257-UUltra-High Performance Fittings for 1/16 in. Tubing

PEEK LiteTouch® Ferrule Assy 1/16 in. (Black) 10 51258-UPEEK Fingertight I Nut (Black) 10-32 10 51262-U PEEK SealTight™ Short Fitting (Black) with Ferrule 10-32 10 51263-UUltra-High Pressure Stainless Steel Fittings

for 1/16 in. Tubing, (with ferrule) 10 51264-Ufor 1/32 in. Tubing, (with ferrule) 10 51265-UUltra-High Pressure Stainless Steel Adapter

for adapting 1/16 in. OD to 1/32 in. OD Tubing 1 51267-UUltra-High Performance MicroTight Unions

for 1/32 in. Tubing PEEK and SS (Fittings & Capsule Union Included) 1 51274-Ufor 360 μm Tubing PEEK and SS 1 51277-Ufor 1/32 in. Tubing PEEK and SS (Fittings Included) 1 51279-UAdapter for 1/16 in. O.D. and 1/32 in. Tubing PEEK and SS 1 51281-UMicroTee

MicroTee PEEK for 1/32 in. for O.D. Tubing 1 51283-UMicroTee PEEK for 360 μm O.D. Tubing 1 51285-UMicroTight® PEEK Tubing Sleeves

MicroTight Sleeve Natural 0.009 I.D. (230 μm) 10 51296-UMicroTight Sleeve Blue 0.011 I.D. (280 μm) 10 51297-UMicroTight Sleeve Natural 0.021.ID. (535 μm) 10 51298-UMicroTight Sleeve Purple 0.006 I.D. (152 μm) 10 51303-UPEEKsil™ Tubing

1/32 O.D. x 25 μm I.D x 50 cm 2 51316-U1/32 O.D. x 50 μm I.D. x 50 cm 2 51321-U1/32 O.D. x 75 μm I.D. x 50 cm 2 51328-U1/32 O.D. x 150 μm I.D. x 50 cm 2 51329-U1/16 O.D. x 50 μm I.D.x 50 cm 2 51332-U1/16 O.D. x 25 μm I.D. x 50 cm 2 51335-U1/16 O.D. x 100 μm I.D. x 50 cm 2 51337-UOPTI-SOLV® Mini Filters

OPTI-SOLV Mini Filter, 0.5 μm 5 51168-UOPTI-SOLV Mini Filter, 2 μm 5 51170-UOPTI-SOLV Micro Filters

OPTI-SOLV Micro Filter, 1.0 μm 5 51172-UOPTI-SOLV Micro Filter, 2.0 μm 5 51173-UOPTI-SOLV Biocompatible Micro Filter, 0.5 μm 5 51174-UOPTI-SOLV Nano Filter

OPTI-SOLV Biocompatible Nano Filter, 0.5 μm 5 51176-UOPTI-GUARD®

OPTI-GUARD 1 mm Guard Column, C18 5 51177-UOPTI-GUARD 1 mm Guard Column, Silica 5 51178-UOPTI-GUARD Biocompatible 1 mm Guard Column, C18 5 51183-UOPTI-GUARD 1 mm Guard Column, C8 5 51184-UOPTI-GUARD 1 mm Guard Column, Amino NH2 5 51187-U OPTI-GUARD 3 mm PEEK/SS Holder 1 51188-UOPTI-GUARD 3 mm Cartridge, C18 3 51191-UOPTI-GUARD 3 mm Cartridge, C8 3 51193-UOPTI-GUARD 3 mm Cartridge, Amino, NH2 3 51194-UOPTI-SOLV EXP® Pre-Column Filter Holder

Holder w / EXP Titanium Hybrid Ferrule, (2 Ferrules, 1 Nut in pack)▼ 1 51163-U▼Note: Cartridges not included with holder.

OPTI-SOLV EXP® Pre-Column Replacement Filter Cartridges

OPTI-SOLV EXP Pre-Column Filter Cartridge, 0.5 μm 5 51164-UOPTI-SOLV EXP Pre-Column Filter Cartridge, 0.5 μm 10 51165-UOPTI-SOLV EXP Pre-Column Filter Cartridge, 0.2 μm 5 51166-UOPTI-SOLV EXP Pre-Column Filter Cartridge, 0.2 μm 10 51167-U

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Reversed-Phase HPLC BuffersHigh quality buffers (solutions, solids or concentrates)

Shyam [email protected]

Preparation of aqueous mobile phase is the most critical

step in reversed-phase chromatography (RPC) method

development for ionic analytes. This includes consider-

ation of the affects of pH on analyte retention, type of

buffer to use, and its concentration, solubility in the

organic modifi er and its affect on detection, among other

considerations. The improper choice of buffer, in terms of

buffering species, ionic strength and pH, can result in

poor or irreproducible retention and tailing in reversed-

phase separation of polar and ionizable compounds.

Problems like partial ionization of the analyte and

strong interaction between analytes and residual silanoles

or other active sites on the stationary phases can be

overcome by proper mobile phase buffering (maintaining

the pH within a narrow range) and choosing the right

ionic species and its concentration (ionic strength) in the

mobile phase (1-2). In sensitive LC-MS separations that

depend heavily on the correct choice of acid, base,

buffering species and other additives (3), a buffer must be

chosen based on its ability to maintain, and not suppress

analyte ionization in the MS interface.

Mobile Phase pH and Reversed-Phase Retention

Retention of ionic analytes in RPC is fundamentally

affected by mobile phase pH. The dissociation properties

of the ionic functional groups also impact analyte

retention. Retention of non-ionic analytes is minimally

affected by mobile phase pH.

For acidic moieties (usually carboxylates), a pH below

the pKa (within limits) and for basic moieties (usually

amines), a pH above the pKa (within limits) of the

compound enhances retention. Dramatic effects are

observed on retention of these analytes in pH range near

the pKa of a given functional group. This becomes

apparent in consideration of chemical dissociation as

illustrated in Figure 1.

The last two equations are commonly known as the

Henderson-Hasselbach equation. These equations suggest

that for pH far removed from the pKa, a small change in the

pH has a minimal affect on the ratio of unprotonated-to-

protonated species. Therefore, moderate change in pH will

not affect retention signifi cantly. However, at pH near the

pKa, a small change in pH will produce a signifi cant change

in the ratio of the two species. Therefore, changing the pH

within a range of values suffi ciently close to the pKa will

dramatically affect retention. When pH is used to increase

reversed-phase retention, the pH should be changed in the

direction that decreases analyte ionization.

Buffer Selection

Buffers are solutions of a weak acid and its conjugate

base, or a weak base and its conjugate acid. They mitigate

the infl uence of hydrogen/hydronium and hydroxide ions

and subsequently reduce the pH fl uctuations, even upon

dilution. The typical pH range for reversed-phase on a

silica-based packing is pH 2 to 8. Choice of buffer is

typically governed by the desired pH. It is important that

the buffer has a pKa close to the desired pH since buffers

control pH best at their pKa. A rule of thumb is to choose

a buffer with a pKa value <2 units of the desired mobile

phase pH (see Table 1).

Phosphoric acid and its sodium or potassium salts are

the most common buffer systems for reversed-phase

HPLC. Phosphate’s two pKa values, 2.1 and 7.1, and UV

transparency make it ideal for most HPLC separations. Its

pKa of 12.3 is suitable for buffering in 11.3-13.3 pH range.

Phosphonate buffers can be replaced with sulfonate

buffers when analyzing organophosphate compounds.

With the growth in popularity of LC-MS, volatile buffer

systems, such as TFA, acetate, formate, and ammonia, are

frequently used.

Buffer Concentration: A higher buffer concentration

that enhance buffer capacity will give more reproducible

separation of compounds partially ionized at the pH of the

HA H+ + A- and Ka =[ H+ ] [ A- ]

HABy defi nition: pH = –log [H+], or pH = pKa + log

The corresponding equation for a weak base is:

or pH = pKa + log[ B ]

[ BH+ ]

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For a weak acid:

[ A- ]

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Table 1. HPLC Buffers, pKa Values and Useful pH Range

sigma-aldrich.com/derivatization

mobile phase, by reducing local perturbations of the pH of

the migrating analyte peak. Generally, a buffer concentra-

tion of 10-50 mM is adequate for small molecules.

Buffer Solubility: It is especially important when

performing gradient separations. Solubility can be

empirically determined by mixing given volume fractions

of buffer and the organic solvent. Appearance of precipi-

tates or opaque solution indicates solubility issues. A

general rule is no more than 50% organic should be used

with a buffer. This will depend on the specifi c buffer as

well as its concentration.

Effects on Detection: The choice of buffer is also

dependent upon means of detection. For traditional UV

detection, the buffer needs to be effectively transparent

in this region, especially critical for gradient separations.

Buffers listed in Table 1 have low enough absorption

below 220 nm.

More common issues today are related to compatibility

with mass spectral (MS) detection. Preferred buffers

addressing the issue of volatility are formate, acetate, and

ammonia. In regard to the issue of suppression of ioniza-

tion, formate and acetate are ideal choices for positive-ion

Featured Products+HPLC-Grade (puris p.a.) Buffers and Additives

Description Pkg. Size Cat. No.

Ammonium acetate, >99.0% (NT) 50 g, 250 g 17836

Ammonium formate, >99.0% (NT) 50 g, 250 g 17843 (dried material)

Ammonium hydroxide solution, 100 mL, 1 L 17837 ~10% (T) in water

Ammonium phosphate monobasic, >99.0% (T) 250 g 17842

Ammonium trifl uoroacetate, >99.0% (NT) 10 g, 50 g 17839

Potassium phosphate dibasic anhydrous, 250 g 17835 >99.0% (T)

Sodium formate, >99.0% (NT) 250 g 17841

Sodium phosphate dibasic dihydrate, ~99% (T) 50 g, 250 g 71633

Sodium phosphate monobasic anhydrous, 50 g, 250 g 17844 >99.0% (T)

Sodium trifl uoroacetate, >99.0% (T) 10 g 17840

Trifl uoroacetic acid:Triethylamine 2M:1M 500 mL 9746

Trifl uoroacetic acid:Triethylamine 2M:2M 100 mL 9747

mode detection. TFA, however, can negatively impact

detector response even in positive-ion mode (4,5), while it

strongly suppresses ionization with negative ion mode.

Acetic acid is good for negative-ion mode. LC-MS applica-

tions further limit buffer selection and buffer concentration.

References 1. McMaster, M.C. HPLC A Practical User’s Guide, VCH Publishers, Inc.: New York,

NY, 1994; 85.

2. Poole, C.F. and Poole, S.K. Chromatography Today, Elsevier Science: Amsterdam, The Netherlands, 1991; 431.

3. Analytix, Five-part series on Mobile Phase Additives for LC-MS, Issue 3, 2006 (www.sigma-ldrich.com/analytix).

4. Temesi, D., Law, B., The Effect of LC Eluent Composition on MS Response Using Electrospray Ionization, LC-GC, 17:626. 1999.

5. Apffel, A. et. al. Enhanced Sensitivity for Peptide Mapping with Electrospray Liquid Chromatography-Mass Spectrometry in the Presence of Signal Suppression Due to Trifl uoroacetic Acid-Containing Mobile Phases, J. Chrom. A. 712:177. 1995.

For a complete list of HPLC buffers and additives, refer

to our online product catalog at: sigma-aldrich.com

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Derivatization Reagents BrochureListing over 400 Derivatization Reagents

To order your free copy either go to sigma-aldrich.com/derivatization, call 800-359-3041 (US and Canada)

or 814-359-3041, email [email protected] or request KDI on the attached card.

● Products sorted by GC, HPLC, Chiral and TLC techniques● Reagents also listed by “Application”● Vials, syringes and other useful items for derivatization reactions● Up-to-date application information and references

Buffer pKa (25°C) Useful pH Range

TFA 0.5 <1.5 Sulfonate 1.8 <1-2.8 Phosphate 2.1 1.1-3.1 Chloroacetate 2.9 1.9-3.9 Formate 3.8 2.8-4.8 Acetate 4.8 3.8-5.8 Sulfonate 6.9 5.9-7.9 Phosphate 7.2 6.2-8.2 Ammonia 9.2 8.2-10.2 Phosphate 12.3 11.3-13.3

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Discovery BIO GFC 3 μm PackingsHillel Brandes and Hugh [email protected]

Gel fi ltration chromatography (GFC) (or size exclusion

chromatography) is a common technique for resolution of

biological macromolecules based on size and/or shape.

Analytes that are large relative to the pore size of the

stationary phase are excluded from the pores and therefore

elute relatively early. Conversely, analytes that are small

relative to the pore size, are well-retained and therefore

elute later. Shape also affects retention: Molecules with a

more elongated structure are not as retained as much as

those that are more spherical or globular.

The Discovery BIO GFC product line for gel fi ltration of

macromolecules is based on uniform, spherical, high-purity

silica with a hydrophilic surface. To date, Discovery BIO GFC

has been available as a 5 μm product line; now, the 3 μm

particles have been added. While the 3 μm will generate

higher backpressures, the relatively low fl ow rates that are

generally optimal for gel fi ltration chromatography only

generate modest back pressures. However, the tremendous

advantage of the 3 μm columns is their higher effi ciency.

This will directly translate to higher resolution and peak

capacity. Request Application Note 187 (T309187) for

further discussion of peak capacity.

Figure 1 compares the elution profi les of a protein

mixture on Discovery BIO GFC, 3 μm versus 5

μm, with the same y-axis scaling. The

improvement in the 3 μm packing is evident

in the narrower and taller peaks. Also evident is

improvements in fi ne detail of the chromato-

gram, which refl ects the higher resolution. A

comparison of the calculated peak effi ciencies,

however, is particularly striking. This is shown

in Table 1 with a sampling of analytes. The

greatest gains are evident with moderately

retained analytes, with peak effi ciencies

improving on the order of 100%. Even for uracil,

representative of a fully retained analyte, effi ciency

has improved 30%, which itself is signifi cant.

Table 1. Peak Effi ciencies (N) of Discovery BIO GFC, 3 μm versus 5 μm

GFC 100 GFC 150 GFC 300Protein/Analyte 5 μm 3 μm % Improvement 5 μm 3 μm % Improvement 5 μm 3 μm % Improvement

BSA monomer -- -- -- 5200 9200 77 10000 19300 93

Ovalbumin -- -- -- 5600 11500 105 9700 18300 89

Myoglobin 13500 24500 81 12600 28800 129 16600 32900 98

Ribonuclease A 14200 25100 77 12900 23400 81 16100 31500 96

Uracil 36400 47900 32 26600 37300 40 26300 36200 38

Figure 1. Elution Profi les of 3 μm Discovery BIO GFC 300 (A) and 5 μm Discovery BIO GFC 300 (B)

column: Discovery BIO GFC 300, 30 cm x 7.8 mm I.D., 3 μm particles (567337-U) mobile phase: 150 mM potassium phosphate monobasic, pH 7.0 (adjusted with potassium hydroxide) fl ow rate: 0.7 mL/min. pressure: 1100 psi temp.: ambient det.: UV, 214 nm injection: 1 μL sample: 1 g/L ea. (except uracil, 0.1 g/L) in mobile phase

1. Thyroglobulin aggregate 2. Thyroglobulin 3. γ-Globulin dimer 4. γ-Globulin monomer

mAU

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6 8 10 12 14 16 18 20mL/min G004834

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in Table 1 w

greatest gain

retained analyt

improving on the

representative of a

has improved 30%

Featured Products+Description Cat. No.

Discovery BIO GFC 100: (100 – 100,000 mw range)

5 cm x 4.6 mm I.D., 3 μm particles 567322-U30 cm x 4.6 mm I.D., 3 μm particles 567323-U5 cm x 7.8 mm I.D., 3 μm particles 567324-U30 cm x 7.8 mm I.D., 3 μm particles 567325-U

Discovery BIO GFC 150: (500 – 150,000 mw range)


Discovery BIO GFC 300: (5,000 – 1,250,000 mw range)


6 8 10 12 14 16 18 20mL/min

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Figure 1. Selectivity of 4 Chiral GC Stationary Phases columns: Supelco β-Dex 110, 30 m x 0.25 mm I.D., 0.25 μm (24301) Supelco β-Dex 120, 30 m x 0.25 mm I.D., 0.25 μm (24304) Astec CHIRALDEX B-DA, 30 m x 0.25 mm I.D., 0.12 μm (72023AST) Astec CHIRALDEX G-TA, 30 m x 0.25 mm I.D., 0.12 μm (7303AST) oven: 60 °C (0 min.), 2 °C/min. to 160 °C (hold 20 min.) inj.: 250 °C det.: FID, 250 °C carrier gas: helium, 25 to 30 cm/sec. set @ 75 °C injection: 1 μL, 100:1 split

sigma-aldrich.com/chiral-chromatography

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Comparing the Enantioselectivity of Cyclodextrin-Based GC Chiral Stationary Phases

Len Sidisky, Kathy Stenerson, Greg [email protected]

Supelco currently offers the widest selection of cyclodex-

trin-based chiral stationary phases. The most versatile and

popular commercial stationary phases are prepared using

alpha-, beta- or gamma-cyclodextrins that have been

functionalized with various derivative groups at the 2, 3

and 6 positions on the sugar molecules. Functional groups

that have been used to derivatize the cyclodextrins include

trifl uoroacetyl, permethyl, dimethyl, diacetyl and others.

Each of the functional groups imparts a unique enantiose-

lectivity on the resulting chiral stationary phase.

We recently compared the selectivity of four popular

chiral GC stationary phases using a series of test probes.

The phases we studied include the Supelco β-DEX™ 110,

β-DEX 120 (both permethylated beta-cyclodextrins imbibed

in an intermediate polarity stationary phase), Astec

CHIRALDEX™ B-DA (a dipentylated beta cyclodextrin) and

Astec CHIRALDEX G-TA (a gamma-cyclodextrin trifl uoro-

acetylated at the 3-hydroxy position and pentylated at the

2- and 6-hydroxy positions).The Supelco β-DEX 110 and

120 and the Astec CHIRALDEX B-DA phases are thought to

interact with chiral compounds primarily using an inclusion

mechanism to affect the separation of the chiral moieties.

The Astec CHIRALDEX G-TA interacts primarily using a

surface interaction with the chiral compounds.

The temperature-programmed analysis of seven chiral

probes shows the Supelco β-DEX 110 and 120 phases

provide similar polarity and chiral selectivity, though there

are differences in their selectivity (Figure 1). These phases

provide chiral resolution primarily via inclusion complex-

ation. The Astec CHIRALDEX B-DA column did not provide

the chiral selectivity for the compounds evaluated compared

to the permethylated and Astec CHIRALDEX G-TA phase.

This phase also uses inclusion complexation as its primary

separation mechanism. The Astec CHIRALDEX G-TA column

had the best chiral selectivity for the compounds used in

this study. The G-TA uses surface interactions as its primary

mechanism to resolve chiral compounds, and has broad

applicability for a variety of chiral compounds.

The Supelco DEX and Astec CHIRALDEX lines comprise

many more phase chemistries than described here. The

columns are also available in convenient and economical

kits containing the most popular phase chemistries. A

partial listing is shown. To view the complete list, please

visit sigma-aldrich.com/chiral-chromatography.


Supelco β-DEX 110, 30 m x 0.25 mm I.D., 0.25 μm 24301

Supelco β-DEX 120, 30 m x 0.25 mm I.D., 0.25 μm 24304

Astec CHIRALDEX B-DA, 30 m x 0.25 mm I.D., 0.12 μm 72023AST

Astec CHIRALDEX G-TA, 30 m x 0.25 mm I.D., 0.12 μm 73033AST

Supelco DEX Kit I 24340 Kit I contents: One 30 m × 0.25 mm I.D., 0.25 μm column of each type: α-DEX 120, β-DEX 120 and γ-DEX 120

Supelco DEX Kit II 24328-U Kit II contents: One 30 m × 0.25 mm I.D., 0.25 μm column of each type: β-DEX 120, β-DEX 225, γ-DEX 225 and β-DEX 325

Astec CHIRALDEX Kit 71030AST Kit contents: One 30 m × 0.25 mm I.D., 0.12 μm column of each type: Astec CHIRALDEX G-TA, B-DM and B-DA

0 10 20 30 40 50Min

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β-DEX 110

β-DEX 120

CHIRALDEX B-DA

CHIRALDEX G-TA

1. Methyl lactate 2. β-Butyrolactone 3. 1-Phenylethanol 4. δ(+), l(-)-Carvone

5. α-Ionone 6. S(+), R(-) Methyl mandelate 7. 1-Octanolactone

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Determination of Triglycerides and Waxes in Food Products Using Cool On-Column Injection and the MET-Biodiesel Capillary Column

Michael D. [email protected]

Introduction

Food nutrition (composition, fat content, labeling

requirements, etc.) and food chemistry (ingredients,

purity, fl avors & fragrances, etc.) are two areas of study in

a food & beverage laboratory. Examples of each are:

● Desired nutritional data includes the triglyceride content of various food products, providing informa-tion that complements that of the fatty acid composition

● Food chemistry analysis includes the characterization of wax content in olive oil, used to help ensure the product is unadulterated

Both of these analyses can be accomplished with the use

of capillary gas chromatography (GC). The suitability of the

MET-Biodiesel capillary column to perform both of these

applications was investigated.

MET-Biodiesel Column

The Supelco MET-Biodiesel capillary GC column was

designed specifi cally for the determination of free and

total glycerin in B100 biodiesel samples. Table 1 lists the

column specifi cations. Several features and benefi ts of this

column indicate that it is also well-suited for the analysis

of triglycerides and waxes in food products. These

features and benefi ts include:

● Metal column holds up better than fused silica, virtually eliminating column breakage

● Integrated guard protects the analytical column, extending column life with a leak-free connection

● Integrated guard also acts as a retention gap, minimizing peak broadening

● Provides good peak shape and resolution for glycerides

● Able to separate mono-, di-, and triglycerides (the mono- and diglycerides analyzed as TMS derivatives)

● Short column length, allowing for fast analysis times ● Operates at a maximum temperature of 380 °C

(isothermal) and 430 °C (programmed)

Our goal was to obtain the acceptable resolution in as

fast of a run time as possible. Several analyses, each using

a different mix, were performed to illustrate the chro-

matographic results that can be obtained with this

column. All separations were performed using cool

on-column injection, which improves the recovery of high

molecular weight substances.

Triglycerides

Triglycerides (also called triacylglycerol, triacylglyceride,

or TAG) are the main constituent of vegetable oil and

animal fat, and make up most of the fats digested by

humans. They are important in that they allow the uptake

and transport of fat-soluble vitamins. Plus, they play a role

in metabolism (unused saturated or monounsaturated

fatty acids are stored by the body as triglycerides).

However, triglyceride intake should be monitored because

high levels of triglycerides have been linked to an in-

creased risk of heart disease and stroke.

Most natural fats contain a complex mixture of indi-

vidual triglycerides. Therefore, effi cient capillary columns

with the ability to provide ample resolution are required

for proper identifi cation. Triglycerides are large com-

pounds requiring a relatively high fi nal oven temperature

for elution in a reasonable time.

Triglycerides in Butter and Lard

The extraction and analysis of triglycerides in butter and

lard samples were performed, with the resulting chro-

matograms shown in Figure 1 and Figure 2, respectively.

Peak IDs follow the nomenclature that T## (Triglyceride

Number or Triacylglycerol Number) signifi es the total

number of carbons on the fatty acid chains, regardless of

degree of saturation or position on the glycerol backbone.

For example, T54 could be SSS, a triglyceride containing

three stearic acids (C18:0). It could also be SOS or SSO, a

triglyceride containing two stearic acids (C18:0) and one

oleic acid (C18:1).

Table 1. MET-Biodiesel Specifi cations Application: This rugged metal column was designed

specifi cally for the determination of free and total glycerin in B100 biodiesel samples. A guard is integrated, thereby providing protection with a leak-free connection (the guard and analytical column are one continuous piece of tubing; there is no union between the guard and analytical column).

USP Code: None Phase: Bonded; proprietary Temperature Limits: -60 °C to 380/430 °C

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The MET-Biodiesel, in combination with cool on-column

injection, fast carrier gas fl ow rates, rapid oven tempera-

ture ramp rates, and high fi nal oven temperatures, was

able to provide signifi cant resolution for both applications

with relatively short run times. As evident, the triglyceride

profi le of butter is much more complex than that of lard.

The profi le differences can be attributed to the fact that

these products are from different sources; butter is

processed from cow milk or cream whereas lard is

rendered from pig fatty tissues.

Glycerides in Palm Oil

The mono-, di-, and triglyceride composition of vegeta-

ble oils can be used as a measurement of quality and purity.

If the oil is adulterated with inferior oil, the peak patterns

and/or ratios will not match up to historical data, poten-

tially falling outside of acceptable control limits.

Because mono- and diglycerides have active hydroxyl

functional groups, they must be derivatized prior to

analysis to minimize analyte adsorption, thereby reducing

peak tailing. An extract of palm oil was analyzed on the

MET-Biodiesel after the mono- and diglycerides were

converted to TMS-derivatives. As shown in Figure 3, the

di- and triglycerides were separated with no overlap

between groups. Additionally, peak shape of the triglycer-

ides is an indication of good column inertness.

Figure 1. Butter Triglycerides Chromatogram courtesy of Dr. M. Povolo and Dr. G. Contarini (CRA-FLC, Lodi, Italy)

column: MET-Biodiesel, 14 m x 0.53 mm I.D., 0.16 μm with integrated 2 m x 0.53 mm I.D. guard (28668-U) oven: 150 °C, 30 °C/min. to 350 °C (15 min.) det.: FID, 400 °C carrier gas: helium, 15 mL/min. injection: 1 μL, cool on-column sample: butter extract

1. T26 2. T28 3. T30


4. T32 5. T34 6. T36

7. T38 8. T40 9. T42

10. T44 11. T46 12. T48

Figure 2. Lard Triglycerides Chromatogram courtesy of Dr. M. Povolo and Dr. G. Contarini (CRA-FLC, Lodi, Italy)

column: MET-Biodiesel, 14 m x 0.53 mm I.D., 0.16 μm with integrated 2 m x 0.53 mm I.D. guard (28668-U) oven: 150 °C, 20 °C/min. to 350 °C (15 min.) det.: FID, 400 °C carrier gas: helium, 15 mL/min. injection: 1 μL, cool on-column sample: lard extract

1. T44 2. T46 3. T48

4. T50 5. T52 6. T54

Figure 3. Palm Oil Glycerides Chromatogram courtesy of Dr. C. Mariani (SSOG, Milan, Italy)

column: MET-Biodiesel, 14 m x 0.53 mm I.D., 0.16 μm with integrated 2 m x 0.53 mm I.D. guard (28668-U) oven: 200 °C, 20 °C/min. to 350 °C (15 min.) det.: FID, 380 °C carrier gas: helium, 10 mL/min. injection: 1 μL, cool on-column sample: palm oil extract (subsequent derivatization with TMS)

1. DAG32 1:2 2. DAG32 1:3 3. DAG34 1:2

4. DAG34 1:3 5. DAG36 1:2 6. DAG36 1:3 7. T48

8. T50 9. T52 10. T54

Triglyceride peak ID nomenclature is the same as discussed

earlier. Diglyceride peak IDs follow the nomenclature that

DAG## (Diacylglycerol Number) signifi es the total number of

carbons on the fatty acid chains, regardless of degree of

saturation. The 1:2 and 1:3 then designate the position of

the fatty acid substitution on the glycerol. For example,

DAG36 1:2 has fatty acids at both the fi rst and second

positions on the glycerol backbone whereas DAG36 1:3 has

fatty acids at both the fi rst and third positions.

Olive Oil

Olive oil is rich in monounsaturated fats, most notably

oleic acid. This is of interest because a diet with a higher

proportion of monounsaturated fats is linked to a

reduction in the risk of coronary heart disease, due to

0 2 4 6 8 10Min G004835

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favorable effects on cholesterol regulation and LDL

cholesterol oxidation, as well as helping to build a more

healthy balance between omega 3 and omega 6 fats.

Therefore, it is desirable to substitute olive oil for other

food products wherever possible.

Olive oil is used throughout the world, especially in the

countries surrounding the Mediterranean Sea. The quality

of the olive oil, infl uenced by the source of the olives and

also how it is processed, determines what it can best be

used for. Possible uses for various grades of olive oils are

shown in Table 2.


Table 2. Grades/Uses of Olive OilGrade UseExtra-virgin olive oil Salads, soups and stews,

dipping breadVirgin olive oil Sautéing ingredientsOlive oil Cosmetics, pharmaceuticals,

soapsOlive-pomace oil Certain kinds of cooking in

restaurantsLampante oil Fuel in traditional oil-burning

lamps (non-edible)

Olive oil is regulated in much of the world to discourage

the labeling of lower grade oils as higher grade oils. As

such, detailed analytical work is performed to fully

characterize the oil. This may include the determination of

free fatty acids, peroxide index, wax content, sterol

content, erythrodiol and uvaol, saturated fatty acids in

position 2 of the triglyceride, spectrophotometric analysis,

fatty acid composition, volatile halogenated solvents,

organoleptic characteristics, stigmastadienes, triglyceride

content, and aliphatic alcohol content.

Olive Oil Waxes

One of the regulated characteristics of olive oil is its wax

content, an indicator of both quality and purity. Because

wax content varies among the various categories of edible

oils, comparing peak patterns and ratios to known

references and specifi cations can determine if the olive oil

has been adulterated with inferior oil.

Because some of these wax compounds may have high

boiling points, GC analysis using a column with a high

temperature limit is desirable. This allows for elution of all

analytes in a reasonable amount of time with good peak

shape. Figure 4 shows the chromatogram obtained from

the analysis of olive oil on the MET-Biodiesel column. The

use of a 350 °C fi nal oven temperature helps to produce

sharp peaks, allowing for easy and accurate peak pattern

and ratio comparisons to known references.

Conclusion

The MET-Biodiesel capillary column is a suitable choice

for analysts who work on triglyceride and/or wax separa-

tions in which high column temperatures are required. The

column’s inertness, high thermal stability, and low bleed

ensure good peak shape and resolution for the analytes

that were evaluated. As shown, this column can be used to

achieve optimal result for these applications.

References 1. ISO/DIS 17678.2, Milk and Milk Products – Determination of Milk Fat Purity by Gas

Chromatographic Analysis of Triglyceride, International Organization for Standard-ization.

2. M. Povolo, V. Pelizzola, and G. Contarini, Directly Resistively Heated Column Gas Chromatography for the Evaluation of Cow Milk Fat Purity, European Journal of Lipid Science and Technology 110.11 (2008) page 1050-1057.

3. Commission Regulation (EEC) No 2568/91 of 11 July 1991, On the Characteristics of Olive Oils and Olive-Residue Oils and on the Relevant Methods of Analysis, Offi cial Journal of the European Communities L 248 (5 September 1991) page 1.

4. Commission Regulation (EC) No 2472/97 of 11 December 1997, Offi cial Journal of the European Communities L 341 (12 December 1997) page 25-39.

5. C. Mariani, P. Bondioli, S. Venturini, and E. Fedeli, Sulla formazione di acidi grassi trans nel processo di raffi nazione dell´olio di oliva lampante, Rivista Italiana delle Sostanze Grasse 68 (1991) page 455-459.

6. M. Buchanan, K. Stenerson, and V. Yearick, Determination of Free and Total Glycerin in B100 Biodiesel, Supelco Reporter 27.1 (2009) page 3-6.

Figure 4. Olive Oil Waxes Chromatogram courtesy of Dr. C. Mariani (SSOG, Milan, Italy)

column: MET-Biodiesel, 14 m x 0.53 mm I.D., 0.16 μm with integrated 2 m x 0.53 mm I.D. guard (28668-U) oven: 200 °C, 20 °C/min. to 350 °C (15 min.) det.: FID, 380 °C carrier gas: helium, 10 mL/min. injection: 1 μL, cool on-column sample: olive oil waxes

1. Diterpenes 2. C40-C46 esters 3. Sterol ester triterpene alcohols

Related Products +Description Cat. No.

Ceramic scribe for cutting capillary columns, Z290254-1PAK pack of 10

Needle-nose pliers, 7½ inch length 22437


MET-Biodiesel, 14 m x 0.53 mm I.D., 0.16 μm 28668-U with integrated 2 m x 0.53 mm I.D. guard

0 2 4 6 8 10 12Min G004836

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SupelMIP SPE – Nitroimidazoles Extraction Procedure

One egg was homogenized. 10 g of the mix was spiked

at levels of 1, 2, and 5 μg/kg nitroimidazoles: DMZ, IPZ,

MNZ, RNZ, and metabolites DMZOH and MNZOH.

Deuterated internal standards of each compound were

added at the spike level of 2 μg/kg. For MNZ and

MNZOH, DMZOH-d3 was used as the internal standard.

10 g of the spiked samples were extracted with 10 mL

acetonitrile followed by centrifugation for 5 minutes. 2 g

sodium chloride was mixed with the isolated supernatant

followed by centrifugation. The extract was evaporated to

dryness in a silanized glass test tube. The residue was

reconstituted in 2 mL DI water and sonicated for 3

minutes prior to SPE processing.

Similar sample pre-treatment was followed for milk

samples; however, the acetonitrile and sodium chloride

extraction process was conducted in one step.

After sample pre-treatment, the resulting samples were

processed using the SupelMIP SPE procedure described in

Figure 2 (page 16).

LC-MS Analysis

After SupelMIP SPE, the samples were analyzed via

LC-MS using the conditions described in Table 1 (page 16).

Relative recovery was determined against internal standards

for each of the spike levels tested. The data was also used

to determine reproducibility and to estimate LLOD. Matrix

(ionization) effects were assessed by spiking nitroimidazoles

into post-SPE blank samples. The results were quantifi ed

against an external calibration curve diluted in buffer.

sigma-aldrich.com/supelmip

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Extraction of Nitroimidazoles from Milk and Eggs using Molecularly Imprinted Polymers

Introduction

Nitroimidazoles are anti-bacterial and anti-coccidial

drugs used in the treatment of cattle, poultry, and pigs.

These compounds and their metabolites are suspected

carcinogens and mutagens. Consequently, their use in

veterinary practice is strictly regulated within European

Union Countries (Council Regulation 2377/90, Annex IV

Banned Compounds).

Metronidazole (MNZ), ipronidazole (IPZ) and ronidazole

(RNZ) belong to a list of pharmacologically active sub-

stances for which no maximum residue limit (MRL) can be

fi xed. As a result, their use is prohibited in food-producing

animals, and any residue of these compounds detected in

animals (and their products) intended for human con-

sumption is considered a violation of EU regulation. The

on-going surveillance of nitroimidazole drug residues and

their metabolites in animal products requires a highly

selective and sensitive assay for trace detection.

In this report, a new molecularly imprinted polymer SPE

phase (SupelMIP™) was evaluated for the extraction of

nitroimidazoles: dimetridazole (DMZ), metronidazole

(MNZ), ipronidazole (IPZ), ronidazole (RNZ), and their

hydroxylated metabolites (DMZOH and MNZOH) (Figure 1).

The nitroimidazole compounds were extracted from milk

and egg samples and analyzed via LC-MS.

What are Molecularly Imprinted Polymers?

Molecularly imprinted polymers (MIPs) are a class of

highly cross-linked polymer-based molecular recogition

elements engineered to bind one specifi c target com-

pound or a class of structurally related compounds with

high selectivity. The MIP material is designed with cavities

that are sterically and chemically complementary to the

target analyte(s). As a result, multiple interactions (e.g.,

hydrogen bonding, ionic, Van der Waals, hydrophobic)

can take place between the MIP captivity and the analyte.

Figure 1. Structure of Nitroimidazoles

G003649 G004839

Dimetridazole (DMZ) Metronidazole (MNZ)

G004840

Ipronidazole (IPZ) Ronidazole (RNZ)


G004841

The following was generated with the assistance of an outside source using Sigma-Aldrich products. Technical content was generated and provided by:

Olga Shimelis1, Anna-Karin Wihlborg2, Marcus Rudolfsson2, Brian Boyd2, and An Trinh1

1. Supelco, Bellefonte, PA, USA2. MIP Technologies AB, Lund, Sweden

Contributed Article

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Figure 2. SupelMIP SPE - Nitroimidazoles Procedure

SPE Column: SupelMIP SPE – Nitroimidazoles, 50 mg/3 mL


High Recoveries, Reproducibility, and Clean Extracts

The relative recovery and RSDs for nitroimidazoles

averaged at 95 ± 7%, and detailed results are described

in Table 2. Representative ion-chromatograms for the

spiked egg samples (1 ng/g) are presented in Figure 3 and

illustrate low background.

Table 1. LC-MS-MS Methodology

Recommended Analytical Technique: LC-MS-MS or LC-MS

column Ascentis C18, 15 cm x 2.1 mm I.D., 3 μm particle size (581302-U) instrument: Sciex API 3200 mobile phase A: 0.1% formic acid in LC-MS grade water mobile phase B: 0.1% formic acid in acetonitrile gradient: Min. A% B% 0.0 95 5 1.0 95 5 8.0 0 10.0 12.0 0 10.0 13.0 95 5 18.0 95 5 fl ow rate: 0.3 mL/min. temp.: ambient det.: MS/MS, MRM transitions DMZ (142/96) IPZOH (186/168) DMZ-d3 (145/99) IPZOH-d3 (189/171) DMZOH (158/140) MNZ (172/128) DMZOH-d3 (161/143) MNZOH (188/126) IPZ (170/124) RNZ (201/140) IPZ-d3 (189/171) RNZ-d3 (204/143) polarity: Positive ion source: Turbospray ion spray voltage: 1200 V source temp: 350 °C collision gas: 4 psi curtain gas: 50 psi inj.: 30 μL

Table 2. % Relative Recoveries (RSDs) of Nitroimida-zoles from Spiked Egg and Milk Samples (n=5)

Sample DMZ1 DMZOH1 IPZ1 MNZ2 MNZOH2 RNZ1

Egg 1 μg/kg 111(7) 106(5) 105(6) 109(3) 83(6) 105(7) Egg 2 μg/kg 84(5) 90(11) 94(5) 91(9) 58(8) 113(20) Egg 5 μg/kg 88(5) 92(8) 90(1) 92(8) 57(13) 134(3) Milk 1 μg/kg 100(8) 1076) 108(6) 105(2) 76(4) 104(3) Milk 2 μg/kg 88(7) 94(3) 97(10) 92(14) 59(7) 111(10) Milk 5 μg/kg 96(10) 101(10) 95(5) 107(9) 62(8) 123(4)

1 Deuterated standard is available, 2 Used DMZOH-d3 as an internal standard

Figure 3. Ion-Chromatograms of 1 ng/g Nitroimidazoles Extracted from Eggs

-500

0

500

1000

1500

2000

-1 1 3 5 7 9 11 13 15

-500

0

500

1000

1500

2000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

-1000

010002000

300040005000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

-2000

200400600800

1000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

-10000

1000200030004000500060007000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

-5000

500100015002000250030003500

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

DMZ

DMZOH

IPZ

MNZOH

RNZ

MNZ

G004791-96

Condition column with:1 mL toluene1 mL acetonitrile1 mL 10 mM ammonium acetate, pH 6.5

Load sample:2 mL sample

Wash: ● 0.5 - 1 mL ultra-pure water

Followed by a strong vacuum through cartridge for 5 min. to remove residual moisture and ensure a dry cartridge (-0.7 bar, -20 in Hg, or –70 kPa)

● 2 x 1 mL hexane ● 1 mL heptane:toluene 3:1 (v/v)

Gentle vacuum (-0.4 bar or –12 in Hg for 5-10 s) between each wash volume

Analyte elution:Elute with 2 x 1 mL acetonitrile:water (60:40, v/v) with 0.5 % acetic acid

Gentle vacuum (-0.4 bar or –12 in Hg for 5-10 s) between each elution volume.

Eluted fraction evaporated to 50 μL at 45 °C under N2 and reconstitute in 500 μL LC-MS mobile phase, fi lter prior to analysis if necessary.

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Conclusion

In this report, SupelMIP SPE – Nitroimidazoles was

evaluated for the extraction of nitroimidazoles and

metabolites from milk and egg. Using molecularly

imprinted polymer technology, high and reproducible

recoveries were observed. Extraction was conducted with

a high degree of selectivity minimizing ion-suppression,

allowing researchers to achieve LODs at the ppt to low

ppb levels. The SupelMIP method described in this report

is highly robust and reproducible offering the necessary

selectivity to achieve the stringent detection limit de-

mands in food safety surveillance.

For more information, please visit our website:


Samples spiked into post-SPE blank extracts revealed

good sample cleanup and low ion-suppression. The matrix

ionization effects were minimal as evident from the

90-120% recovery values depicted in Table 3.

Table 3. Determination of Matrix Effects – Absolute Recovery of Post-SPE Spiked Samples

Sample DMZ DMZOH IPZ MNZ MNZOH RNZ

Egg 91 92 103 96 99 89 Milk 118 110 123 116 98 109

Table 4. Estimated LOD of Nitroimidazoles using SupelMIP SPE

LOD (ng/g) RNZ MNZ DNZ DMZOH MNZOH IPZ

Egg 0.016 0.020 0.066 0.124 0.061 0.026 Milk 0.010 0.023 0.063 0.162 0.076 0.015

Featured Products+Description Oty. Cat. No.

SupelMIP SPE - Nitroimidazoles, 50 mg/3 mL 50 52734-U

Ascentis C18 Column, 15 cm x 2.1 mm I.D., 1 581302-U 3 μm particle size

The estimated detection levels of nitroimidazoles ranged

from 0.010-0.16 μg/kg in milk and 0.016-0.12 μg/kg in egg

(Table 4). The LODs were estimated by analyzing the

analyte and background response levels of the lowest

spike concentration; and extrapolating a theoretic

signal-to-noise ratio of 3:1.

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The Analysis of Resveratrol in Red Wine by On-Fiber Derivatization/SPME

Katherine [email protected]

Introduction

Resveratrol is a phytoallexin produced by grapes and

other plants to increase resistance to fungal infection.

Recent research suggests that consumption of resveratrol

may reduce the risk of certain cancers, heart disease, and

other age-related disorders (1). Red wine, which is produced

by fermentation of juice on the crushed grapes, has been

found to contain a greater amount of resveratrol than white

wine, which is produced by fermentation of the juice alone.

In this study, the extraction and analysis of resveratrol

from red wine is demonstrated using solid phase microex-

traction (SPME) and GC-MS. The presence of 3-hydroxyl

(OH) groups make it necessary to derivatize this compound

prior to GC analysis. Derivatization was conducted, after

extraction, directly on the SPME fi ber by exposing it to the

vapors of a silyating reagent. The derivative of resveratrol

was then analyzed directly from the fi ber using GC-MS.

Experimental

The extraction and derivatization conditions used are

summarized in Table 1. The polyacrylate fi ber, which is

suitable for the extraction of polar semivolatiles and is more

resistant to swelling than other types of fi bers, was chosen

based on recently published fi ndings (2,3). The extraction

conditions were based on these works as well, with some

modifi cations made to extraction and desorption times to

decrease matrix interference from the wine sample.

minutes directly in the injection port of the GC-MS. The

derivatization procedure resulted in silylation of all three

–OH groups present in resveratrol (Figure 1). The resulting

derivative has a molecular weight of 444, and subsequent

GC-MS analysis showed a predominance of the molecular

ion, which was used for quantifi cation.

Calibration standards ranging from 10 – 300 μg/L were

prepared by spiking a solution of 12% ethanol in water

with trans resveratrol. Red wine (California merlot),

diluted 3:1 in 12% ethanol in water, was analyzed both

“as is” and spiked with a known level of trans-resveratrol.

Results/Discussion

A plot of the calibration standards as absolute response

vs. concentration is presented in Figure 2. Linearity was very

good, with a correlation coeffi cient of 0.9987, indicating

that the extraction and derivatization procedure is quantita-

tive. Peak responses obtained using the technique were

adequate to allow for detection of the lowest calibration

standard while using the MSD in full scan mode.

Chromatograms of the unspiked and spiked red wine

samples are presented in Figures 3 and 4. Using the

average response factor from the calibration, the levels of

trans-resveratrol in each were calculated, along with a

percent accuracy of this experimental value relative to the

known spiking level of the wine sample (Table 2).

Table 1. SPME – On-Fiber Derivatization Conditions sample/matrix: 3 mL of red wine (California merlot) diluted 3:1 in 12% ethanol:water SPME fi ber: 85 μm polyacrylate extraction: immersion at room temperature, 15 min., with stirring at 400 rpm derivatization: 20 min. in 4 mL vial containing 5 μL of Sylon-BFT desorption: 280 °C, 2 minutes

Figure 1. Derivatization of Resveratrol

G004842

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After sample extraction, the SPME fi ber was gently

blotted with a Kimwipe® to remove excess water. The fi ber

was then inserted into a 4 mL vial containing 5 μL of Sylon™

-BFT (BSTFA + 1% TMCS) reagent. For consistency, the vial

containing the Sylon-BFT was allowed to equilibrate for 60

– 90 minutes prior to use, and a new reagent vial was used

for each extraction. The fi ber was desorbed at 280 °C for 2

Table 2. Wine Spike Levels Unspiked Spiked Red Wine Red Wine (100 μg/L)

Conc. of trans-resveratrol (μg/L) 22.6 134.7

% Accuracy --- 110%

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Conclusions

SPME when used in combination with on-fi ber

derivatization was found to be applicable to the extrac-

tion of resveratrol from red wine. The technique was

found to be highly sensitive, simple, and quantitative.

The polyacrylate fi ber was also found to withstand

exposure to the vapors of the silylating reagent without

damage resulting from swelling.

References 1. Red Wine Compound Resveratrol Demonstrates Signifi cant Health Benefi ts.

ScienceDaily. Retrieved June 18, 2009, from http://www.sciencedaily.com /releases/2009/06/090611174052.htm

2. Cai, Lingshuang, Koziel, Jacek A., Dharmadhikari, Murli, van Leeuwen, J. (Hans), Rapid Determination of trans-resveratrol in red wine by solid-phase microextrac-tion with on-fi ber derivatization and multidimensional gas chromatography-mass spectrometry. J. Chrom. A (2009), 1216, 281-287.

3. Vinas, Pilar, Campillo, Natalia, Martinez-Castillo, Nelson, Hernandez-Cordoba, Manuel, Solid-phase microextraction on-fi ber derivatization for the analysis of some polyphenols in wine and grapes using gas chromatography-mass spectrometry. J. Chrom A (2009), 1216, 1279-1284.

Featured Products+Description Qty. Cat. No.

SPME Fibers

Manual, w/ 85 μm polyacrylate coating 3 57304

Auto, w/ 85 μm polyacrylate coating 3 57305

Auto, 23 GA w/ 85 μm polyacrylate coating 3 57294-U

GC Column

Fused silica capillary column, SLB-5ms, 28471-U 30 m x 0.25 mm I.D., 0.25 μm

Chemical Standards and Reagents

Resveratrol 100 mg R5010

BSTFA + TMCS, 99:1 (Sylon BFT) 20 x 1 mL 33148 25 mL 33155-U 50 mL 33149-U

For more information on SPME products and applications request a copy of the 7th edition SPME applications CD, T199925 (CJQ), or visit sigma-aldrich.com/spme.

Related Information!Figure 4. SPME On-Fiber Derivatization Analysis of Resveratrol in Spiked Red Wine SampleIdentical conditions to those in Figure 3

Figure 2. SPME Calibration Standards; 10-300 μg/LSPME - on-fi ber derivatization analysis of trans-resveratrol

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peak

are

a (x

1,0

00,0

00)

35

30

25

20

15

10

5

00 50 100 150 200 250 300 350

Conc. (μg/L) G004843

R2 = 0.9987

Figure 3. SPME On-Fiber Derivatization Analysis of Resveratrol in Unspiked Red Wine Sample

column: SLB-5ms, 30 m x 0.25 mm I.D., 0.25 μm (28471-U) oven: 100 °C (1 min.), 10 °C/min. to 325 °C (3 min.) MSD interface: 325 °C scan range: m/z 40-450 carrier gas: helium, 1 mL/min, constant liner: 0.75 mm I.D. SPME

6 8 10 12 14 16 18 20 22 24Min G004844

trans-resveratrol (TMS)

cis-resveratrol (TMS)

6 8 10 12 14 16 18 20 22 24Min G004845

trans-resveratrol (TMS)

cis-resveratrol (TMS)

In addition to the trans form, a sizable cis-resveratrol

peak was detected in the wine samples. The cis form is not

naturally found in grapes, however it has been theorized

that it can be formed from the trans during analysis, or the

production and/or aging of wine (2).

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Sigma-Aldrich Expands Environmental Product Line by Offering NSI Solutions Inorganic Products

Vicki [email protected]

Sigma-Aldrich Corporation and NSI Solutions, Inc.

(Raleigh, NC) have partnered to provide US customers

with a more complete selection of quality analytical

products for environmental testing from a single vendor.

NSI is an ISO registered, A2LA accredited provider of

inorganic and microbiological profi ciency testing samples,

as well as quality control standards (QC) for oil and

grease, turbidity, solids, and pH analyses.

These products can be ordered by calling Customer

Service at 800-811-5352, or by visiting our special

Sigma-Aldrich Vendor Portal located on the website:

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Microbiology Profi ciency Testing (PT) Samples

These microbiological PT samples are designed for use

with all Most Probable Number (MPN) and Membrane

Filter (MF) procedures. All PT and QC samples are

packaged with a cold pack to maintain integrity and are

drop shipped to preserve sample traceability.

Description Cat. No.

WS Microbiology Profi ciency Testing

WS - Microbiological PT NSI-MIC-001 Each set includes 10 samples and 10 vials of sterile diluent. Works with EPA promulgated methods for total coliform, fecal coliform and E. coli

WS - Standards Plate Count NSI-MIC-002 One vitroid sample containing a heterotrophic bacteria. Supplied with sterile diluent.

WS Quantitative Coliforms NSI-MIC-006 One vitroid sample in the range of 20-200 CFU/100mL, designed for LT2 Enhanced Surface Water Treatment Rule. Evaluated for total coliform, fecal coliform, and E. coli. Applicable for all SDWA quantitative methods. Supplied with hydration fl uid.

Microbiological QC Known Standards

Drinking Water Set - Total and Fecal Coliform NSI-MIC-QC1 Works with EPA approved methods. Each set contains one vitroid sample each of the following: total/fecal coliform positive, total coliform positive/fecal coliform negative, one total fecal and coliform negative, and one blank sample. Each set includes 4 x 100 mL bottles of sterile diluent.

Standards Plate Count NSI-MIC-QC3 Designed for use with SM9215-B Pour Plate Method. Set contains a single quantitative sample of heterotrophic bacteria and a 100 mL bottle of sterile diluent.

WS - Microbiological QC NSI-MIC-QC4 Each set includes 10 samples and 10 vials sterile diluent. Works with EPA promulgated methods for total coliform, fecal coliform, and E. coli.

WS Quantitative Coliforms NSI-MIC-QC6 One vitroid sample in the range of 20 -200 CFU/100mL, designed for LT2 Enhanced Surface Water Treatment Rule. Evaluated for total coliform, fecal coliform, and E. coli. Applicable for all SDWA quantitative methods. Supplied with hydration fl uid.

Oil & Grease Standards for US EPA Method 1664

NSI Oil and Grease PAR

(Precision and Recovery)

Surrogate SNIPS are pack-

aged in exact volumes in

easy-to-use inert, thermo

sealed PTFE tubes. Simply

snip off the top of the

tube and pour the entire

contents into your extraction vessel. Also available are

the more conventional bottles of oil and grease stan-

dards as well as recovery standards.

Products are supplied with a certifi cate of traceability

listing certifi ed concentrations and acceptance limits.


Method 1664 Oil and Grease PAR Surrogate (0.2%) 0.2% n-hexadecane, 0.2% stearic acid in acetone delivers 40 mg total oil & grease per tube 25 x 10 tubes NSI-QC-003LSNIP

Method 1664 Oil and Grease PAR Surrogate (0.4%) 0.4% n-hexadecane, 0.4% stearic acid in acetone delivers 80 mg total oil & grease per tube 25 x 10 tubes NSI-QC-003LSNIP

Oil and Grease, Ready-to-Use Bottles Works with freon and hexane extraction. Oil and grease supplied as 20-100 mg/L. Minimum of 10 lots available per year. 500 mL NSI-QCI-069 4 x 500 mL NSI-QCI-069C

Oil and Grease, Supplied in Boston Round Bottle Designed for use with SPE. Oil and grease supplied as 20-100 mg/L. Minimum of 10 lots available per year. 1 Liter NSI-QCI-069-33,400 4 x 1Liter NSI-QCI-069-33,400C

Oil and Grease Method 1664 Recovery Standards (0.4%) 25 mL NSI-QC-003

0.4%(w/v) n-Hexadecane and 0.4% (w/v) Stearic acid in acetone 10 x 25 mL NSI-QC-003TP

Oil and Grease Method 1664 Recovery Standards (0.2%) 25 mL NSI-QC-0003L

0.2%(w/v) n-Hexadecane and 0.2% (w/v) Stearic acid in acetone 10 x 25 mL NSI-QC-003LTP

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Turbidity QC Check StandardsThese Turbidity Check Standard Solutions are ideal

second-source check standards. The products are

traceable to formazin, ready-to-use as received, and

applicable to all turbidimeters. Products are supplied with

certifi cates of traceability listing certifi ed concentrations

and acceptance limits.


100 NTU* 500 mL NSI-QCI-152 4 x 500 mL NSI-QCI-152C 3.78 Liters NSI-QCI-152L

50 NTU 500 mL NSI-QCI-153 4 x 500 mL NSI-QCI-153C 3.78 Liters NSI-QCI-153L




* NTU = Nephelometric Turbidity Units

Solids Standards & Concentrates, pH Standards

Mid and high level solids

standards are formulated to

contain Total Dissolved

Solids (TDS), Total Suspend-

ed Solids (TSS), and Total

Solids (TS). They are stable

at room temperature,

non-hazardous, and

easy-to-use. Simply shake

the bottle well and measure

an appropriately sized aliquot for use. No pipeting,

diluting or formulating is needed. The economical 4 x 1

liter case will provide up to 250 QC analyses for the high

level concentrations.

The low level TSS standard is as easy-to-use as the mid

and high level standards and is specifi cally formulated for

Minimum Detectable Limit (MDL) validations.

These products are traceable to NIST, compliant with

SM 1020 B, Section 3, and value priced (1). A certifi cate of

traceability listing certifi ed concentrations and acceptance

limits is shipped with each product.


Low Level TSS Standard 4 x 1 Liter NSI-QCI-057 TSS 15-30 mg/L

Mid-Level Solids Standard 4 x 1 Liter NSI-QCI-055 TDS 500-1500 mg/L, TS 500-1600 mg/L, TSS 60-100 mg/L

High-Level Solids Standard 4 x 1 Liter NSI-QCI-055H TDS 1000-3000 mg/L, TS 1400-3500 mg/L, TSS 400-500 mg/L

pH QC Standard 4 x 500 mL NSI-QCI-112C pH 5-10 units, NIST traceable with multiple lots always available.

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Inorganic Water Supply (WS) and Non-Potable Water (NPW) PT Samples

No sample is released into a PT study unless the results

meet NSI’s acceptance limits, which is 30% more stringent

than the NELAC standards. The purity of each analyte

source material is certifi ed, and then the PT sample

assigned value is corrected for this purity. The accuracy of

each formulation and the homogeneity of each batch are

validated by instrumental analyses for each analyte in each

of the nine samples taken from the production run.

All PT samples are supplied in duplicate and are drop

shipped to maintain product traceability. All shipments

include instructions for storage and preparation, as well

as report forms.

Pkg. PT Samples QC KnownDescription Size Cat. No. Cat. No.

WS (Drinking Water) PT Samples

WS-Hardness 250 mL PEI-145 NSI-QCI-145 For determination of: Calcium 30-90 mg/L Calcium Hardness 75-375 mg/L Magnesium 2.0-20 mg/L Total Hardness 83-307 mg/L Sodium 12-24 mg/L

WS-Inorganic 500 mL PEI-041 NSI-QCI-041 For determination of: Chloride 5-100 mg/L Sulfate 5-500 mg/L Fluoride 1-8 mg/L Alkalinity 25-200 mg/L Nitrate as N 3-10 mg/L Conductivity 250-2500 umhos/cm Nitrate/Nitrite-N 3.5-9.0 mg/L Tot. Dissolved Solids 200-450 mg/L Potassium 10-40 mg/L

WS-pH 250 mL PEI-083 NSI-QCI-083 For determination of pH without dilution. Formulated in the NELAC range of 5.0-10.0 units.

WS-Metals 2 x 21 mL PEI-016 NSI-QCI-016 Each ampul produces 2 liters of sample. For determination of the following elements: Aluminum 130-2500 μg/L Lead 5-100 μg/L Antimony 6-50 μg/L Manganese 40-900 μg/L Arsenic 5-50 μg/L Molybdenum 15-130 μg/L Barium 500-3000 μg/L Nickel 10-500 μg/L Beryllium 1-10 μg/L, Selenium 10-100 μg/L Boron 800-2000 μg/L Silver 20-300 μg/L Cadmium 2-50 μg/L Thallium 2-10 μg/L Chromium 10-200 μg/L Vanadium 315-2500 μg/L Copper 50-2000 μg/L Zinc 400-2500 μg/L Iron 100-1800 μg/L

WS-Nitrite 21 mL PEI-140 NSI-QCI-140 Concentrate. Each ampul produces 2 liters of sample. Formulated in the NELAC range of 0.40-2.00 mg/L.

WS-Orthophosphate 21 mL PEI-141 NSI-QCI-141 Concentrate. Formulated in the NELAC range of 0.5-5.5 mg/L.

WS-Residual Free Chlorine 0.5 mL PEI-012 NSI-QCI-012 Concentrate for determination of residual free chlorine and total residual chlorine. Each ampul produces 2 liters of sample. Formulated in the NELAC range of 0.500-3.00 mg/L.

NPW (Wastewater) PT Samples

NPW-Demands 21 mL PEI-026 NSI-QCI-026 Concentrate. Each ampul produces 2 liters of sample.

NPW-Minerals 500 mL PEI-136 NSI-QCI-136 Ready-to-use sample packaged in an HDPE bottle to be analyzed for: Chloride 35-275 mg/L Alkalinity 10-120 mg/L Fluoride 0.3-4 mg/L Conductivity 200-930 umhos/cm Potassium 4.0-40 mg/L TDS at 180 °C 140-650 mg/L Sodium 6-100 mg/L Sulfate 5-125 mg/L

NPW-Hardness 250 mL PEI-137 NSI-QCI-137 Ready-to-use sample packaged in an HDPE bottle to be analyzed for: Calcium 3.5-110 mg/L Calcium Hardness 8.7-275 mg/L Magnesium 2.0-40 mg/L Total Hardness 17-440 mg/L

NPW-pH 250 mL PEI-035 NSI-QCI-035 For pH analysis without dilution. Formulated in the NELAC range of 5.0-10.0 units.


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NPW-Residue 500 mL PEI-079 NSI-QCI-079 Ready-to-use whole volume sample to be analyzed for total suspended solids in the NELAC range of 23-100 mg/L and total solids formulated in the NELAC range of 140-675 mg/L.

NPW-Simple Nutrients 21 mL PEI-138 NSI-QCI-138 Concentrate. Each ampul produces 2 liters of sample. Ammonia as N 0.65-19.0 mg/L Nitrate/Nitrite as N 0.25-40.0 mg/L Nitrate as N 0.25-40.0 mg/L Orthophosphate as P 0.50-5.50 mg/L

NPW Complex Nutrients 21 mL PEI-139 NSI-QCI-139 Concentrate. Each ampul produces 2 liters of sample. TKN 1.50-35.0 mg/L Total Phosphorus 0.50-10.0 mg/L

NPW Trace Metals 2 x 21 mL PEI-034 NSI-QCI-034 Each ampul produces 2 liters of sample. For analysis of the following elements: Aluminum 200-4000 μg/L Manganese 70-4000 μg/L Antimony 95-900 μg/L Molybdenum 60-600 μg/L Arsenic 70-900 μg/L Nickel 80-3000 μg/L Barium 100-2500 μg/L Selenium 90-2000 μg/L Beryllium 8-900 μg/L Silver 26-600 μg/L Cadmium 8-750 μg/L Strontium 30-300 μg/L Chromium 17-1000 μg/L Thallium 60-900 μg/L Cobalt 28-1000 μg/L Tin 1000-5000 μg/L Copper 40-900 μg/L Titanium 80-300 μg/L Iron 200-4000 μg/L Vanadium 55-2000 μg/L Lead 70-3000 μg/L Zinc 100-2000 μg/L

NPW-Mercury 21 mL PEI-087 NSI-QCI-087 A 21 mL concentrate for determination of Mercury. Each ampul produces 2 liters of sample. Formulated in the NELAC range of 2.0-30 μg/L.

NPW Total Residual Chlorine 2.2 mL PEI-033 NSI-QCI-033 Concentrate. Each ampul produces 2 liters of sample. Formulated in the NELAC range of 0.50-3.00 mg/L.

Visit sigma-aldrich.com/nsisolutions for a complete

list of NSI products now available through Sigma-Aldrich.

Reference 1. American Public Health Association. Standard Methods for the Examination of

Water & Wastewater. 20th ed.; Baltimore, MD, 1999.

Characterized Reference Oils for Standardizing Lipid Procedures

We offer characterized reference oil samples for use

as controls or check samples for fatty acid methyl ester

(FAME) analyses. These samples provide an excellent

means of standardizing your lipid procedures and

comparing your results to other researchers. Each

standard is packaged in an amber ampul under

nitrogen. Package size is 1000 mg. A Certifi cate of

Composition is provided with each oil sample.

Description CAS No. Cat. No.

Canola oil 120962-03-0 46961Coconut oil 8001-31-8 46949Corn oil 8001-30-7 47112-UCottonseed oil 8001-29-4 47113Lard oil 8016-28-2 47115-ULinseed (fl axseed) oil 8001-26-1 47559-UMenhaden fi sh oil 8002-50-4 47116Olive oil 8001-25-0 47118Palm oil 8002-75-3 46962Peanut oil 8002-03-7 47119Saffl ower oil 8001-23-8 47120-USoybean oil 8001-22-7 47122Sunfl ower seed oil 8001-21-6 47123

Mono-, Di-, andTriglyceride Chemical Standards

Sigma-Aldrich offers numerous qualitative mono-, di-,

and triglyceride standards for determining relative retention

times and establishing approximate response factors when

used in chromatographic applications. These standard

mixtures are prepared by weight, and the composition

verifi ed by gas and/or thin layer liquid chromatography. A

certifi cate of composition is provided with each standard.

A sampling of this product line is listed below. Addi-

tional glyceride standards can be found by visiting

sigma-aldrich.com.

Glyceride and Phospholipid MixturesDescription Pkg. Size Cat. No.

Mono-, Di-, & Triglyceride Mix 40 mg 1787-1AMP 1,3-Diolein, 10 mg Monoolein, 10 mg 1,2-Dioleoyl-rac-glycerol, 10 mg Triolein, 10 mg

Triglyceride Mixtures 100 mg 17811-1AMP Tricaprin, ~20 mg Tripalmitin, ~20 mg Tricaprylin, ~20 mg Trimyristin, ~20 mg Trilaurin, ~20 mg

Phospholipid Mixture for HPLC 2 mL P3817-1VL derived from soybeans, in chloroform (varied conc.) L-α-Lysophosphatidylcholine, 0.3 mg/mL L-α-Phosphatidylcholine, 1.5 mg/mL L-α-Phosphatidylethanolamine Escherichia coli, 1.2 mg/mL L-α-Phosphatidylinositol ammonium salt from Glycine max (soybean), 0.9 mg/mL

Glyceride KitsDescription Cat. No.

Mono-, Di-, and Triglycerides Kit MDT12-1KT (individually packaged in 100 mg each) Dilaurin 1-Monopalmitoyl-rac-glycerol Dimyristin 1-Monostearoyl-rac-glycerol Dipalmitin Trilaurin Distearin Trimyristan 1-Monolauroyl-rac-glycerol Tripalmitin 1-Monomyristoyl-rac-glycerol Tristearin

Triglycerides Kit TRI19-1KT (individually packaged in 100 mg unless otherwise indicated) Triacetin Trilaurin Triarachidin Trilinolein Tribehenin Trilinolenin Tributyrin Trimyristin Tricaprin Triolein Tricaproin, 1 mL Tripalmitin Tricaprylin, 0.5 mL Tripalmitolein Trielaidin Tripetroselinin Tri-11-eicosenoin Tristearin Trierucin

Saturated, Even Carbon Triglycerides Kit TRI11-1KT (individually packaged in 100 mg unless otherwise indicated) Triacetin Tricaprylin, 1 mL Triarachidin Trilaurin Tribehenin Trimyristin Tributyrin Tripalmitin Tricaprin Tristearin Tricaproin, 1 mL


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Featured Products+Slim Line (6.5 mm O.D. syringe barrel), 50 mm Cone Tip Needle SGE Syringes for CTC CombiPAL and GC PAL

Volume Description Needle Pack Size SGE Cat. No. Supelco Cat. No.

Slim Line, Fixed Needle

5 μL 5F-C/F-5/0.63 23 gauge (0.63 mm OD) 1 001981 29622-U 5 μL 5F-C/F-5/0.47 26 gauge (0.47 mm OD) 1 001982 29623-U 10 μL 10F-C/F-/0.63 23 gauge (0.63 mm OD) 1 002981 29624-U 10 μL 10F-C/F-5/0.47C 26 gauge (0.47 mm OD) 1 002980 29625-U 25 μL 25F-C/F-0.47C 26 gauge (0.47 mm OD) 1 003980 29628-U

Slim Line, Fixed Needle, Gas Tight

10 μL SK 10F-C/F-5/0.47C 26 gauge (0.47 mm OD) 6 002986 29626-U 10 μL 10F-C/F-GT-0.63 23 gauge (0.63 mm OD) 1 002987 29627-U 25 μL 25F-C/F-GT-0.63 23 gauge (0.63 mm OD) 1 003987 29629-U 100 μL 100F-C/F-GT-0.63 23 gauge (0.63 mm OD) 1 005335 29631-U 100 μL 100R-C/F-GT-0.47C 26 gauge (0.47 mm OD) 1 005333 29632-U

Slim Line, Removable Needle

0.5 μL 0.5BNR-C/F-0.63 23 gauge (0.63 mm OD) 1 000492 29633-U 10 μL 10R-C/F-5/0.47C 26 gauge (0.47 mm OD) 1 002982 29634-U 10 μL 10R-C/F-0.63 23 gauge (0.63 mm OD) 1 002984 29635-U

Slim Line, Removable Needle, Gas Tight

10 μL 10R-C/F-GT-0.47 26 gauge (0.47 mm OD) 1 002985 29636-U

sigma-aldrich.com/ctc-gc-syringes

Acc

esso

ries

SGE Slim Line Syringes for CTC/Leap Autosamplers

A gas chromatograph (GC) autosampler syringe serves

as the interface between the sample and the instrument;

compatibility is critical for rapid and precise sampling. The

wrong sized syringe can lead to inconsistent results and,

more seriously, can damage the instrument. Syringes also

must deliver accurate amounts with no-dead volume or

carryover between injections.

Sigma-Aldrich has introduced a new line of GC

autosampler syringes from SGE™ that are made to be used

with CTC CombiPAL™ and GC PAL autosamplers. SGE Slim

Line syringes are designed with a unique plunger that

eliminates bending and seizing and are tested to ensure

optimal fi t within autosampler specifi cations. The cone tip

point style of the needle withstands multiple and fast

septum injections, making it one of the most trouble-free

autosampler syringes available.

When used with CombiPAL and GC PAL autosamplers,

these syringes are installed into a syringe holder designed

to accommodate the outside diameter of the syringe barrel.

SGE Slim Line autosampler syringes are available with

both fi xed and removable needle with the following

specifi cations:

● Volumes from 0.5 to 100 μL ● Needle length of 50 mm ● Point style 1 (cone tip) ● Two gauge sizes: 23 (0.63 mm) and

26 (0.47 mm) O.D.

For more information on any of our syringes, email our

Technical Service Department at [email protected]

or visit us on the web at sigma-aldrich.com/syringes.

TRADEMARKS: Ascentis, CHIRALDEX, DEX, Discovery, HybridSPE, Sigma-Aldrich, Supelco, SupelMIP, Supelclean, Sylon – Sigma-Aldrich Biotechnology LP; CombiPAL – CTC Analytics; Fused-Core – Advanced Materials Technologies, Inc.; Kimwipe – Kimberly-Clark Corporation; LiteTouch, MicroTight, SealTight – IDEX Health and Science, Inc (Upchurch Scientifi c); OPTI-SOLV, OPTI-GUARD, EXP – Optimize Technologies, Inc.; SGE – SGE Analytical Science; PEEKsil – SGE International Pty. Ltd.; Tefzel – E.I. du Pont de Nemours and Company

E001072

E001073

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Success through Innovation and

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©2009 Sigma-Aldrich Co. All rights reserved. SIGMA, , SAFC, , SIGMA-ALDRICH, ALDRICH, , FLUKA, , and SUPELCO, are trademarks belonging to Sigma-Aldrich Co. and its affiliate Sigma-Aldrich Biotechnology, L.P. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip.

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Supelco Reporter Vol. 27 - Sigma-Aldrich